Ap20187

The iCasp9-NK cells along with unmodified NK cells were incubated for 24 h in a medium, with the addition of: PBS, DMSO, and 100 nM of chemical inductor of dimerization (CID) (AP20187 (MedChemExpress, Monmouth Junction, NJ, USA). DMSO was taken at a concentration corresponding to its content in a sample with 100 nM CID. The level of apoptosis and cell viability were measured by flow cytometry with the use of AnnexinV-AF647 (Invitrogen, San Jose, CA, USA) and Sytox-VioBlue (Invitrogen, San Jose, CA, USA).

Ganciclovir (Genentech, San Francisco, CA; 25 mg/kg), dissolved in water and phosphate‐buffered saline [PBS]) or PBS alone, was administered i.p. for five consecutive days, with 10–14 days between the last dose of one treatment round and the first day of the next treatment round. AP20187 (MedchemExpress, Monmouth Junction, NJ; 10mg/kg, dissolved in ethanol for stock solution, then diluted in PEG‐400 and 2% Tween for dosing solution) or PEG‐400/Tween 2% in water (vehicle) was administered by i.p. injection for three consecutive days, with 12 days between treatments. Dasatinib (LC Laboratories, Woburn, MA; 5 mg/kg) and quercetin (Sigma Aldrich, St. Louis, MO; 50 mg/kg) or vehicle (60% Phosal, 10% ethanol, and 30% PEG‐400) were administered for five consecutive days monthly via oral gavage in cohort 1, or for three consecutive days, with 14 days between the last dose of one treatment round and the first day of the next treatment round in cohort 2. For CyTOF experiments, a single course of AP20187 (3 days) or D + Q (5 days) was given 3 days prior to sacrifice. Navitoclax (Active Biochem, Kowloon, Hong Kong; 50 mg/kg) or vehicle (60% Phosal, 10% ethanol, and 30% PEG‐400) was administered via oral gavage for five consecutive days monthly. Body composition was measured by EchoMRI (Echo Medical Systems, Houston, TX).

Unless otherwise specified, reagents were obtained from Millipore Sigma. Palbociclib (#HY-50767A), and AP20187 (#HY-13992) were purchased from MedChem Express. Human mammary adenocarcinoma MCF7 cells (RRID:CVCL_0031) and triple negative breast carcinoma (TNBC) MDA-MB-231 (RRID: CVCL_0062) cells, as well as mouse mammary adenocarcinoma TS/A cells (RRID:CVCL_VQ63) were kindly provided by Dr. Sandra Demaria (Weill Cornell Medicine). All cell lines were routinely maintained at 37 °C under 5% CO2, in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5 mM L-glutamine, 5 mM HEPES buffer, 50 μM β-mercaptoethanol 100 U mL⁻¹ penicillin sodium, 100 µg mL⁻¹ streptomycin sulfate and 50 µg mL⁻¹ gentamycin. Cells were authenticated by STR profiling (a service provided by IDEXX Bioresearch) and periodically checked for Mycoplasma spp. contamination by the PCR-based LookOut® Mycoplasma PCR Detection Kit. All cells were employed for experiments 2–10 passages after thawing. All irradiation procedures were performed on a Small Animal Radiation Research Platform (SARRP, from Xstrahl).